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Abstract

Members of the IL-1 family play protective and regulatory roles in immune defense against the opportunistic mold Aspergillus fumigatus. In this study, we investigated the IL-1 family member IL-33 in lung defense against A. fumigatus. IL-33 was detected in the naive lung, which further increased after exposure to A. fumigatus in a dectin-1–independent manner. Mice deficient in the receptor for IL-33 (Il1rl1−/−) unexpectedly demonstrated enhanced lung clearance of A. fumigatus. IL-33 functioned as a negative regulator of multiple inflammatory cytokines, as IL-1α, IL-1β, IL-6, IL-17A, and IL-22 were significantly elevated in fungal-exposed Il1rl1−/− mice. Subsequently, IL-33 administration to normal mice attenuated fungal-induced IL-17A and IL-22, but not IL-1α, IL-1β, or IL-6, production. IL-33–mediated regulation of IL-17A and IL-22 did not involve the modulation of IL-23 but rather PGE2; PGE2 was significantly increased in fungal-exposed Il1rl1−/− mice, and normal mice produced less PGE2 after fungal exposure when administered IL-33, suggesting that IL-33–mediated regulation of IL-17A and IL-22 occurred at the level of PGE2. This was confirmed by in vivo cyclooxygenase 2 inhibition, which attenuated fungal-induced IL-17A and IL-22, as well as IL-1α, IL-1β, and IL-6, production in Il1rl1−/− mice, resulting in impaired fungal clearance. We also show that a PGE2 receptor agonist increased, whereas a PGE2 synthase inhibitor decreased, the levels of IL-17A and IL-22 but not IL-1α, IL-1β, or IL-6. This study establishes novel mechanisms of innate IL-17A/IL-22 production via PGE2 and regulation of the PGE2/IL-17A/IL-22 axis via IL-33 signaling during lung fungal exposure.

Introduction

Concerns over the rise in invasive fungal infections (IFIs) over the last several decades as the result of modern medical interventions (new, more effective immunosuppressive drugs/regimens) are mounting (1). IFIs caused by Aspergillus fumigatus remain one of the most lethal human infectious diseases. Invasive aspergillosis (IA) is particularly insidious as the result of a combination of increasing antifungal drug resistance, extremely high mortality, and an inherent difficulty in diagnosis (2, 3). Although IA is the most devastating aspect of A. fumigatus exposure and accounts for 20–40% of IFIs in solid organ and hematopoietic transplantation (4, 5), colonization with, or sensitization to, A. fumigatus has dramatic effects on lung function in asthmatics (6) and individuals with cystic fibrosis (7).

The IL-1 family of cytokines is composed of 11 members: 7 with proinflammatory characteristics (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ) and 4 with anti-inflammatory characteristics (IL-1Ra, IL-36Ra, IL-37, and IL-38). Early studies in experimental models of A. fumigatus infection documented the induction of IL-1 family members, such as IL-1β (8, 9). We have further shown that the fungal β-glucan receptor dectin-1 drives IL-1α and IL-1β production in vivo during IA (10). A recent study has demonstrated that IL-1R was essential for survival, with IL-1α promoting leukocyte recruitment and IL-1β promoting antifungal activity (11). Studies in humans have shown that A. fumigatus strongly induces IL-36γ and IL-36Ra, but not IL-36α, which were dependent on dectin-1 and TLR4 (12). Inhibiting IL-36 signaling was found to abrogate the induction of protective Th17 and Th1 responses. With respect to immunopathogenic effects of IL-1 family members during IA, IL-1α and IL-1β are thought to be the primary drivers of inflammation in chronic granulomatous disease via decreased autophagy and increased inflammasome activation (13). Finally, analysis of polymorphisms within the IL-1 family revealed that a single nucleotide polymorphism in IL-1Ra VNTR2 (rs380092), IL-1α-889C (rs1800587), and IL-1β (rs1143627) correlates with the incidence or risk for developing IA in hematological patients (14).

IL-33 is recognized as 1 of 11 members of the IL-1 cytokine family because of its chromosome cluster, structure, and processing (reviewed in Ref. 15). IL-33 has been studied in a variety of inflammatory conditions, including allergic asthma, colitis, rheumatoid arthritis, and cardiovascular diseases (reviewed in Ref. 16). Likewise, IL-33 has been investigated in a variety of viral (influenza, respiratory syncytial virus), bacterial (sepsis, Gram-negative keratitis, Gram-positive skin infection), and fungal (Candida albicans peritonitis, fungal asthma) infection models (17). By and large, IL-33 mediates the induction of type 2/Th2 responses and suppression of Th1 and inflammatory responses. However, IL-33 has the potential to mediate a diverse array of functions, such as neutrophil recruitment and reactive oxygen species/reactive nitrogen intermediates induction, depending on the site and type of insult (17). Although IL-33 has yet to be investigated in IA, blockade of IL-33R signaling has been shown to improve fungal asthma severity, putatively through the modulation of proallergic responses (18). In the current study, we defined the role of IL-33 in lung defense against IA. We show that IL-33 is detrimental for lung antifungal defense as a result of its function as a negative regulator of IL-17A and IL-22 via suppression of cyclooxygenase 2 (COX-2)/PGE2.

Materials and Methods

Mice

Wild-type (WT) C57BL/6 (BL/6) mice, 6–8 wk of age, were obtained from The Jackson Laboratory (Bar Harbor, ME) or Taconic (Hudson, NY). Il1rl1−/− (ST2/IL-33R) mice were a kind gift from Dr. A. McKenzie (Cambridge University). Il1r1−/− mice were obtained from The Jackson Laboratory. All animals were housed in a specific pathogen–free, Association for Assessment and Accreditation of Laboratory Animal Care–certified facility and handled according to Public Health Service Office of Laboratory Animal Welfare policies after review by the University of Alabama at Birmingham Institutional Animal Care and Use Committee.

A. fumigatus isolate 13073 (American Type Culture Collection, Manassas, VA) was maintained on potato dextrose agar for 5–7 d at 37°C. Conidia were harvested by washing the culture flask with 50 ml of sterile PBS supplemented with 0.1% Tween 20. The conidia were passed through a sterile 40-μm nylon membrane to remove hyphal fragments and enumerated on a hemocytometer. For challenge, mice were lightly anesthetized with isoflurane and administered 7 × 107A. fumigatus conidia in a volume of 50 μl intratracheally, as previously described (10, 19). Briefly, mice are held in a vertical upright position, and the tongue is withdrawn from the mouth using forceps. A pipette is used to deliver the 50-μl inoculum to the caudal oropharynx, and normal breathing results in fluid aspiration into the lungs (20). For lung fungal burden analysis, the left lungs were collected at 48 h postexposure and homogenized in 1 ml of PBS. Total RNA was extracted from 0.1 ml of unclarified lung homogenate using the MasterPure Yeast RNA Purification Kit (Epicentre Biotechnologies, Madison, WI), which includes a DNase treatment step to eliminate genomic DNA, as previously reported (21). Total RNA was also extracted from serial 1:10 dilutions of live A. fumigatus conidia (101–109) and treated with DNase to form a standard curve. Lung A. fumigatus burden was analyzed with real-time PCR measurement of A. fumigatus 18S rRNA [GenBank accession number AB008401 (22)] and quantified using a standard curve of A. fumigatus conidia, as previously described (21). As a validation of the real-time PCR method, heat-killed A. fumigatus did not yield a signal by real-time PCR and were unable to grow on potato dextrose agar plates (21). In addition, no-amplification controls (i.e., no reverse transcriptase included in the cDNA reaction) yielded a signal < 0.001% by real-time PCR, indicating that the DNase treatment step efficiently eliminated contaminating A. fumigatus DNA [because DNA is not predicative of organism viability (23)].

In vitro treatments (PGE2 inhibition, EP receptor agonist)

In specific experiments, WT BL/6 mice were challenged with A. fumigatus, and lungs were collected and enzymatically digested 48 h later. Cells were enumerated and plated at 1 × 106 cells in a volume of 0.2 ml in the presence of vehicle, an inhibitor of microsomal PGE synthase (mPGES)-1 (Cayman Chemical), or misoprostol, a global EP receptor (PGE2 receptor) agonist (Sigma). Supernatants were collected after 24 h and clarified by centrifugation, and inflammatory cytokine and IL-22 levels were quantified by Bio-Plex and ELISA (R&D Systems), respectively (10).

Statistics

Data were analyzed using GraphPad Prism Version 5.0 statistical software (GraphPad, San Diego, CA). Comparisons between groups were made with the two-tailed unpaired Student t test when data were normally distributed. Significance was accepted at a p value <0.05.

Results

IL-33 is detrimental for lung infection with the Th1-requiring fungal pathogen Cryptococcus neoformans as a result of its known propensity to induce type 2/Th2 responses (25). IL-33 is protective in a model of C. albicans peritoneal sepsis via the induction of heightened neutrophil responses (26). Elimination of A. fumigatus from the lung is highly dependent on neutrophils (27). Moreover, there is evidence to suggest that innate type 2 responses may also be beneficial in A. fumigatus lung clearance (28). Collectively, host immune responses initiated by IL-33 would appear to favor A. fumigatus lung clearance. In initial studies, we demonstrate that IL-33 is increased in response to acute A. fumigatus challenge (Fig. 1A); however, unlike our previous report on fungal asthma (29), IL-33 did not require dectin-1–mediated β-glucan recognition for induction (Fig. 1A). To our surprise, mice deficient in IL-33R (ST2/T1/IL1-RL1; Il1rl1−/−) demonstrated lower A. fumigatus burden in the lung 48 h postchallenge (Fig. 1B), suggesting that IL-33 induction functions as a negative regulator of fungal clearance. We did not observe a difference in fungal burden prior to 48 h postchallenge (data not shown). Intriguingly, augmented fungal clearance in Il1rl1−/− mice at 48 h postchallenge was not a consequence of differential lung cellularity (Fig. 1C), because we observed no significant changes in neutrophils and eosinophils (gated on CD11b+ cells, followed by Ly6G+ cells as neutrophils and Siglec-F+ cells as eosinophils) (Fig. 1D) or inflammatory monocytes (gated on CD11b+ Ly6C+ cells, followed by gating on CCR2+ cells) (Fig. 1E) (all of which have been implicated in innate lung clearance of A. fumigatus) (10, 30, 31). Similar data were observed at 12 and 24 h postinfection (Supplemental Fig. 1). This observation is supported by no differences in the levels of various chemokines produced by lung digest cells (Fig. 1F) and similar levels of inflammatory cells and inflammation in H&E-stained lung tissue sections (Fig. 1G). Sequential Grocott's methenamine silver–stained lung tissue sections also demonstrated lower levels of A. fumigatus organisms in Il1rl1−/− mice (right panel) compared with WT mice (left panel) (Fig. 1H). Thus, despite the induction of IL-33 after acute A. fumigatus exposure, clearance of the organism from the lung is enhanced in the absence of IL-33 signaling.

Because we did not observe major cellular changes in the lung between A. fumigatus–exposed WT and Il1rl1−/− mice, we examined whether there was a difference in inflammatory factors that could explain improved fungal clearance in the absence of IL-33 signaling. We observed significantly higher levels of IL-1α, IL-1β, and IL-6, a profile that favors the development of IL-17A and IL-22, which were also produced at higher levels by lung digest cells from Il1rl1−/− mice (Fig. 2A). Higher inflammatory cytokine levels in the absence of IL-33 signaling suggested that IL-33 functioned as a negative regulator of these mediators. To confirm this, we challenged mice with A. fumigatus, followed by treatment with IL-33, which resulted in an ∼65% reduction in IL-17A (Fig. 2B) and an ∼50% reduction in IL-22 production (Fig. 2C). Although this suggests that IL-33 treatment could have a negative effect on the clearance of A. fumigatus from the lung, we did not observe an effect of IL-33 treatment on fungal burden (Fig. 2D). Thus, the absence of IL-1RL1/ST2 signaling results in augmented lung clearance of A. fumigatus in the presence of heightened IL-17A and IL-22 production, indicating that signaling through IL-1RL1 serves as a negative regulator of these mediators.

PGE2, but not IL-23, is increased in the absence of IL-33 signaling

IL-23 is the most recognized positive regulator of IL-17A and IL-22 production and we have previously reported that IL-23 drives IL-17A and IL-22 production in the lung after A. fumigatus exposure (19, 24). Therefore, we speculated that the increased production of IL-17A and IL-22 in the absence of IL-33 signaling was most likely the result of increased IL-23. However, Il1rl1−/− mice had similar levels of IL-23 in the lung as WT mice (Fig. 3A). There also was no difference in IL-23 production by A. fumigatus–stimulated bone marrow–derived dendritic cells between WT and Il1rl1−/− mice (Supplemental Fig. 2). These data suggested that IL-17A and IL-22 were elevated in Il1rl1−/− mice via an IL-23–independent mechanism. PGE2 has recently been described as a potentiator of innate IL-22 production (32); therefore, we questioned whether PGE2 was modulated in the absence of IL-33 signaling. Results show that, at baseline, lung cells from naive WT and Il1rl1−/− mice produced similar levels of PGE2 (Fig. 3B); however, upon exposure to A. fumigatus, lung cells from Il1rl1−/− mice demonstrated significantly higher PGE2 production (Fig. 3B). These data suggest that IL-33 signaling regulates PGE2 production during A. fumigatus exposure. We next examined mice treated with IL-33 after A. fumigatus exposure and found that, like IL-17A and IL-22, PGE2 levels were significantly lower (Fig. 3C). Thus, IL-33 is a negative regulator of PGE2, which may function as a positive regulator of IL-17A and IL-22 production during lung fungal exposure.

PGE2 supports production of IL-17A and IL-22, but not IL-6, IL-1α, or IL-1β, by lung cells from A. fumigatus–exposed mice

Although PGE2 is a product of COX-2 signaling, it is unclear whether PGE2 itself affects inflammatory mediator production. To address this, we cultured lung cells from infected mice with an inhibitor of mPGES-1, which converts the COX-2 product PGH2 into the biologically active PGE2. This demonstrated that specific inhibition of PGE2 attenuated IL-17A and IL-22 production (Fig. 5A) but not that of IL-6, IL-1α, or IL-1β (Fig. 5B). Conversely, treating lung cells from infected mice with the global EP receptor (PGE2 receptor) agonist misoprostol increased IL-6, IL-17A, and IL-22 production (Fig. 5C) over vehicle-treated lung cells, but it did not increase production of IL-1α and IL-1β (Fig. 5D). Misoprostol was not effective at inducing these mediators in lung cells from naive mice (Supplemental Fig. 3). Collectively, our data reveal that PGE2 specifically promotes the production of IL-17A and IL-22 during lung fungal exposure.

PGE2 supports IL-17A and IL-22, but not IL-6, IL-1α, or IL-1β, production by lung cells from A. fumigatus–exposed mice. (A and B) BL/6 WT and Il1rl1−/− (IL-1RL1; ST2-deficient) mice were challenged with A. fumigatus conidia; at 48 h after exposure, mice were sacrificed, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured in the presence of the mPGES inhibitor (I) or vehicle (V; DMSO) in triplicate for 24 h. IL-17A and IL-22 levels (A) and IL-6, IL-1α, and IL-1β levels (B) in coculture supernatants were quantified by Bio-Plex or ELISA. The figure illustrates cumulative data from three independent studies (n = 1 or 2 mice per group, per study). (C and D) BL/6 WT mice were exposed, and cells were isolated as in (A). Cells were cultured in the presence of misoprostol or vehicle in triplicate for 24 h. IL-6, IL-17A, and IL-22 levels (C) and IL-1α and IL-1β levels (D) in coculture supernatants were quantified by Bio-Plex or ELISA. The figure illustrates cumulative data from two independent studies (n = 1 or 2 mice per group, per study). *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student t test.

Increased IL-1α and IL-1β signaling is not the mechanism associated with enhanced PGE2 and IL-22 production in Il1rl1−/− mice after A. fumigatus exposure

Thus far, data indicate that the level of PGE2 directly correlates with the level of IL-17A and IL-22 after A. fumigatus exposure; however, it is not clear why PGE2 is elevated in Il1rl1−/− mice after A. fumigatus exposure. In addition to IL-17A and IL-22, data in Fig. 2A showed that IL-1α and IL-1β were significantly elevated in Il1rl1−/− mice after A. fumigatus exposure. IL-1β has previously been implicated in PGE2 induction (33). In turn, mice deficient in the receptor for IL-1α and IL-1β demonstrated an ∼60% reduction in PGE2 levels (Fig. 6A), as well as a profound reduction in the production of IL-22 (Fig. 6B), suggesting that the increase in IL-1α and/or IL-1β in the absence of IL-33 signaling was potentially responsible for the increase in PGE2 and, thus, IL-22 production. To confirm this, we determined whether IL-1α and IL-1β levels were modulated in mice that received IL-33 after A. fumigatus challenge. Despite the fact that IL-33 signaling impaired PGE2 (Fig. 3C) and IL-22 (Fig. 2C) production, IL-33 administration had no effect on the levels of IL-1α or IL-1β (Fig. 6C). There was also no synergy between IL-1β and PGE2 receptor stimulation, because the addition of IL-1β, in conjunction with misoprostol, to lung cells from A. fumigatus–infected mice did not augment production of IL-22 (Supplemental Fig. 3). Thus, the mechanism behind elevated PGE2 in the absence of IL-33 signaling is not a result of enhanced IL-1α and IL-1β production.

Increased IL-1R signaling is not the mechanism associated with enhanced PGE2 and IL-22 in Il1rl1−/− mice after A. fumigatus exposure. BL/6 WT and Il1r1−/− (IL-1R1–deficient) mice were challenged intratracheally with A. fumigatus conidia, and 48 h after exposure, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured in triplicate for 24 h. PGE2 levels were quantified in clarified coculture supernatants by enzyme immunoassay (A), and IL-22 levels were quantified in clarified coculture supernatants by ELISA (B). The figures illustrate cumulative data from four independent studies (n = 1 or 2 mice per group, per study). (C) BL/6 WT mice were challenged with A. fumigatus and administered IL-33 (1 μg in 50 μl) or PBS intratracheally 6 and 24 h later. Forty-eight hours after exposure, the right lungs were collected and enzymatically digested, and unfractionated lung cells were cultured in triplicate for 24 h. IL-1α and IL-1β levels were quantified in clarified coculture supernatants by Bio-Plex. The figure illustrates cumulative data from two independent studies (n = 1 or 2 mice per group, per study). ***p < 0.001, unpaired two-tailed Student t test.

Discussion

In this article, we demonstrate that, despite the induction of IL-33 in the lung in response to A. fumigatus, IL-33 signaling negatively regulated immune responsiveness to A. fumigatus, as evidenced by mice deficient in IL-33R signaling (ST2; Il1rl1−/− mice) displaying lower fungal burden postchallenge. Assessment of potential mechanisms to explain enhanced fungal clearance in the absence of IL-33 signaling revealed a specific profile of elevated cytokines: IL-1α, IL-1β, IL-6, IL-17A, and IL-22. Despite observing elevated levels of these mediators, they did not correlate with any changes in lung cellularity, an observation itself supported by the lack of changes in the levels of multiple chemokines. As a result, we focused on the IL-17A/IL-22 axis, because of their known abilities to induce soluble antimicrobial responses from the lung epithelium (34) and our previous work documenting a protective role for IL-17A and IL-22 in host defense against A. fumigatus (19, 24). An intriguing finding from the current study was that IL-33 functioned as a negative regulator of IL-17A and IL-22. Relatively few cytokines have been identified as negative regulators of IL-17A and IL-22. The most well-recognized include TGF-β, which promotes IRF4-mediated suppression of IL-17A and IL-22 (35). Others include IL-27, which promotes c-Maf–mediated inhibition of IL-17A and IL-22, and IL-25, which favors type 2 cytokine induction and, consequently, IL-17A and IL-22 suppression. Overall, our findings suggest the addition of IL-33 to this list, because the absence of IL-1RL1/ST2 signaling results in heightened IL-17A and IL-22 production, whereas increasing IL-33 levels results in decreased IL-17A and IL-22 production, indicating that IL-33 signaling through IL-1RL1 serves as a negative regulator of IL-17A and IL-22.

Based on our previous work (19, 24), our hypothesis behind elevated IL-17A and IL-22 levels in the absence of IL-33 signaling was that IL-33 negatively regulated IL-23, which we have shown to drive IL-17A and IL-22 production in the lung (19, 24). However, this was found not to be the case, resulting in a search for an IL-23–independent mechanism of enhanced IL-17A and IL-22 production. A role for PGE2 in the generation of IL-17A and IL-22 has been controversial. It has been reported that PGE2 may enhance IL-17A production from previously polarized Th17 cells or memory Th17 cells (36, 37); however, recent data show that PGE2 has the complete opposite (i.e., negative) effect on Th17 generation from naive T cells (38). In this latter study, the fungal pathogen C. neoformans was shown to produce its own PGE2, which negatively affected IL-17A production but had no effect on IL-22 (38). Other studies showed that anti-CD3 activation of human PBMCs in the presence of exogenous PGE2 promoted IL-17A production but significantly dampened IL-22 production (39). Human PBMCs stimulated with the fungus C. albicans exhibited increased IL-17A and IL-22 production that was slightly affected by the NSAID diclofenac (p < 0.05 for IL-17A; IL-22 was slightly lower but not statistically significant) (40). In this report, EP receptor agonists were moderately effective at restoring NSAID-inhibited IL-17A production, although the effects on IL-22 were not determined. However, as described earlier, a recent report demonstrated that the COX2/PGE2 axis functioned in the generation of innate IL-22 production in humans and mice (32). In this study, using an experimental model of LPS-induced systemic inflammation, PGE2–EP4 signaling promoted homeostasis of type 3 innate lymphoid cells and drove them to produce IL-22. We found that, similar to IL-17A and IL-22, PGE2 levels were elevated in Il1rl1−/− mice; treating mice with IL-33 resulted in the lowering of IL-17A, IL-22, and PGE2 levels in the lung. Although IL-17A and IL-22 levels were reduced after IL-33 treatment (approximately two thirds and one half, respectively), they were not completely eliminated. Moreover, IL-33 treatment had no effect on the levels of IL-1α, IL-1β, or IL-6. Consequently, we did not observe an effect of IL-33 treatment on A. fumigatus lung clearance. Based on these results, we hypothesize that IL-33 treatment does not facilitate a level of immunosuppression that compromises fungal clearance. In contrast, when targeting PGE2 via COX-2 inhibition, IL-17A and IL-22 levels, as well as IL-1α, IL-1β and IL-6 levels, in Il1rl1−/− mice were significantly reduced, which correlated with reduced A. fumigatus lung clearance. An unexpected finding from our work was that neutralization of IL-17A and IL-22 in Il1rl1−/− mice did not impair A. fumigatus lung clearance. Based on observations with IL-33 treatment (reduced IL-17A and IL-22 levels; intact IL-1α, IL-1β, and IL-6 levels; and no effect on fungal clearance) and celecoxib/Celebrex treatment (reduced IL-17A, IL-22, IL-1α, IL-1β, and IL-6 levels and a negative effect on fungal clearance), the mechanism of resistance in Il1rl1−/− mice appears to be quite complex. Specifically, intact (IL-33 treatment) or elevated (Il1rl1−/− mice) IL-1α, IL-1β, and/or IL-6 may compensate in some manner when IL-17A and IL-22 levels are manipulated. It would be of interest in future studies to cross Il1rl1−/− mice with Il1r−/− mice, which would eliminate IL-1α and IL-1β signaling in tandem with IL-33 signaling and may result in mice deficient in IL-17A and IL-22 induction, as well as IL-1α and IL-1β signaling.

When targeting PGE2 via COX-2 inhibition, IL-17A and IL-22 levels, as well as IL-1α, IL-1β, and IL-6 levels, in Il1rl1−/− mice were significantly reduced, further suggesting that COX-2–mediated PGE2 contributed in some way to the heighted IL-17A and IL-22 production observed in these mice. To confirm that PGE2 had the specific ability to induce IL-17A and IL-22, addition of a PGE2 agonist to lung cells from A. fumigatus–exposed mice increased IL-6, IL-17A, and IL-22 production but not IL-1α or IL-1β. Conversely, specific inhibition of PGE2 had a negative effect on IL-17A and IL-22 production from lung cells, but not IL-1α, IL-1β, or IL-6 production. The novelty in our findings is not only that the COX-2/PGE2 axis drives innate IL-17A and IL-22 production in a mucosal tissue (i.e., the lung), suggesting a conserved mechanism of IL-17A and IL-22 induction, it also identifies putative upstream regulators of the PGE2/IL-17A/IL-22 axis (i.e., IL-33 signaling). A question arising from our findings is how does IL-33 signaling regulate PGE2 induction and, thus, control the level of inflammatory cytokines during fungal exposure? IL-1 signaling was a prime candidate responsible for increased PGE2 in Il1rl1−/− mice, because IL-1α and IL-1β were elevated in these mice after fungal exposure, and IL-1 signaling is known to induce PGE2 (41, 42). However, despite a profound reduction in IL-17A and IL-22 levels, IL-1R deficiency resulted in only a moderate reduction in PGE2 production. Moreover, treating mice with IL-33 did not attenuate the levels of IL-1α and IL-1β, despite significantly lower PGE2 levels. Collectively, these results suggest that other IL-1–independent factors were responsible for elevated PGE2 levels in Il1rl1−/− mice. To this end, we assessed additional factors that were increased in A. fumigatus–exposed Il1rl1−/− mice, yet were decreased in mice that were treated with IL-33. This analysis identified the cytokines IL-3, IL-17A, and IFN-γ. Although published reports suggest that each of these may induce PGE2 (43–47), neutralization of IL-3, IL-17A, or IFN-γ in lung cell cultures from Il1rl1−/− mice did not result in attenuated PGE2 or IL-22 production (Supplemental Fig. 4). Based on these observations, we hypothesize that a soluble factor, such as a cytokine, is an unlikely candidate for heightened PGE2 production in the absence of IL-33 signaling. IL-1RL1 (and, thus, IL-33) has been shown to regulate TLR2 responses, because macrophages from Il1rl1−/− mice show hyperresponsiveness when stimulated with bacterial lipoprotein (48). Because we have previously reported a small role for TLR2 in alveolar macrophage responses to A. fumigatus (49), it is possible that enhanced TLR2 signaling in Il1rl1−/− mice contributes to higher PGE2 and IL-17A and IL-22 induction in some manner. Indeed, fungal recognition via TLR2 can drive PGE2 production (50). To this end, we cannot exclude the possibility that IL-33 may affect the expression or function of other pattern recognition receptors (i.e., the β-glucan receptor dectin-1). Another study has shown that macrophages from mice deficient in Atf3, an ATF/CREB basic leucine zipper transcription factor induced during cellular stress, have enhanced COX-2 signaling and PGE2 production after stimulation with zymosan (51). Ongoing studies are interrogating the role of IL-33 in Atf3 induction, as well as its effect on dectin-1.

In conclusion, we have identified a novel role for IL-33 in the regulation of IL-17A and IL-22 production in the lung after fungal exposure. The absence of IL-33 signaling resulted in enhanced IL-17A and IL-22 production in the presence of enhanced PGE2 levels, whereas the opposite was true in mice administered IL-33. Inhibition of COX-2 signaling in vivo resulted in lower IL-17A and IL-22 induction and impaired fungal clearance. A specific agonist of PGE2 receptors augmented, whereas specific inhibition of PGE2 generation impaired, IL-17A and IL-22 production. Overall, the current body of work adds depth to our understanding of the role that specific IL-1 family members play in IL-17A and IL-22 regulation and innate lung defense. Moreover, these findings lay the foundation for determining whether IL-33 signaling affects PGE2 and/or IL-17A and IL-22 induction in other IL-17A/IL-22–dependent models of inflammation, such as colitis and psoriasis, or infection models requiring IL-17A/IL-22 for defense, such as Gram-negative bacterial pneumonia and influenza.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

This work was supported by Public Health Service Grants HL096702, HL122426, and HL136211.

. 1982. Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes.J. Clin. Invest.69: 617–631.